During the previous year, this laboratory identified the elusive coreceptors mediating HIV entry into CD4+ target cells. The molecules are chemokine receptors, members of the superfamily of G protein-coupled receptors. Initially two distinct coreceptors were identified: fusin (subsequently renamed CXCR4) which functions preferentially for T-cell line-adapted HIV-1 strains, and CCR5 which functions preferentially for macrophage-tropic strains. Our studies during the present year have focused on: 1) Role of these and other coreceptors in HIV entry, transmission, and pathogenesis. Genetically diverse primary HIV-1 strains were found to use various combinations of CCR5 and CXCR4, as well as CCR3; a close correlation was observed between the ability of an Env to use specific coreceptors and its ability to mediate fusion with natural CD4+ target cells expressing endogenous coreceptors. A newly isolated chemokine receptor-like protein was also found to function as coreceptor for HIV-1, and also for SIV. A CCR5 allele containing a 32 base-pair deletion was detected at high frequencies in Caucasian populations (20% heterozygous, 1% homozygous). The truncated protein encoded by this allele displays no coreceptor activity. Epidemiological analyses indicated that individuals homozygous for the CCR5 deletion allele are markedly resistant to HIV infection, consistent with the fact that macrophage-tropic isolates are preferentially transmitted between individuals. CCR5 and CXCR4 expression were found to be regulated differently in primary T cells, and the expression pattern depends greatly on the mode of T cell activation; these results have important implications for immune reconstitution protocols, and may also shed light on mechanisms underlying the natural evolution of the HIV quasispecies in the infected person. 2) Mechanism of coreceptors in fusion/infection. Coreceptor activity was found not to require G protein signaling. Two mechanisms were delineated for chemokine inhibition of HIV entry: coreceptor downmodulation and direct blocking. Studies with chimeric coreceptors and with coreceptor-based synthetic peptides revealed that different classes of HIV Envs use different extracellular regions of a given coreceptor; the transmembrane and/or cytoplsmic regions were also shown to be important, possibly by influencing conformation of the extracellular regions. Soluble CD4 was shown to activate Env for fusion with cells bearing coreceptors but no CD4. 3) Therapeutic strategies based on the coreceptors. Studies have been initiated to screen combinatorial libraries to identify small molecules that bind to the coreceptors and inhibit HIV infection.